Education

From Pathways Databases to Network Models

of Switching Behavior
Baltazar D. Aguda*, Andrew B. Goryachev

Introduction

resources on the Internet. Note that we are not concerned

here with computational methods of determining or
inferring network topology, or connectivity from omics data
on this topic the reader is referred to an article of Qi and Ge
[3] that appeared in this journal recently.
In this article, we illustrate how one extracts a reduced
network model from a large preliminary network obtained
from databases. The model extraction procedure is explained
in the context of a specic biological question about a cell
cycle checkpoint called the restriction point (R point)
that is, what is the smallest subset of interactions in the given
network that can account for the switching behavior
associated with this checkpoint? A method of qualitative
network analysis is proposed to zoom into a core subnetwork
which accounts for the essential qualitative behavior being
modeled. Once the core network model is established, a
kinetic model is constructed and a suite of mathematical
analysis and computer programs can be used for further
investigation.

The excitement in todays biology is driven by the huge

amounts of information generated by high-throughput dataacquisition technologies, and by the expectation that these
datasets will soon provide detailed understanding of lifes
processes. Ultimately, these datasets have to be integrated
into a framework that facilitates the study of the dynamics
arising from networks of physicochemical interactions
orchestrating the physiology of a biological cell. The
bioinformatics community is actively responding to this call
for integration in terms of frameworks of pathways databases
[1,2]. This paper addresses the use of these databases as
sources of dynamical models for biological phenomena. We
focus here on models that are based on molecular
interactions and how these interactions are coupled to
explain observed cellular behavior. The model-building
process that we describe below takes the point of view of a
non-biologist who has access to online pathways databases
but has not been directly involved in relevant experimental
studies. Of course, one could argue that a better approach is
for the modeler to collaborate with a biologist who is already
familiar with the system and has developed intuition about
how it works; in other words, the biologist may already have a
model in mindusually called a hypothesisand what
remains to be done is to encode this model in the language of
mathematics. Note that this hypothesis-driven modeling
approach already assumes a reduced network in the
beginning of the modeling process. In contrast, we would like
to show in this paper how a reduced network model is
extracted from a much larger network, given a specic
biological question and a set of relevant experimental
observations.
Mathematical models range from qualitative and
probabilistic models to quantitative and deterministic kinetic
models [35]. The chosen set of molecular interactions and
processes form what we call a network model. Although the
networks can have various degrees of detail, they all have the
common property of being composed of nodes and edges
representing interactions between nodes. Denitions of
networks and pathways, as well as an example of a network
model are given in the next section. Ultimately, we are
interested in mechanistic models with well-dened molecular
interactions or reaction mechanisms and corresponding rate
equations that are subsequently solved numerically to
simulate the phenomenon. Although mechanistic details are
becoming available in increasing numbers of online pathways
databases and knowledgebases, quantitative values of most
kinetic parameters are still lackingand this problem is
compounded by the fact that many details of these pathways
can be cell-specic (with regard to cell type, organism, etc.)
and can have variability even among the same cell type in an
organism. We provide below an overview of pathway
PLoS Computational Biology | www.ploscompbiol.org

Qualitative versus Mechanistic Network Models

We limit our denition of a network to a connected graph
composed of nodes and edges. An edge connects at most two
nodes, and the connectedness of the graph means that there
exists at least one continuous path (regardless of edge
direction) linking any two nodes in the network. A node can
represent physical entities (genes, ions, molecules, protein
complexes, etc.) or modules (dened to include processes or
subnetworks with identiable functions, but whose internal
details are not shown in the graph; for example, the
mechanistic details of a signaling module can be hidden if we
are merely interested in the modules inputoutput response
to external signals). Thus, a qualitative network model
presumes that the set of nodes and edges is sufcient to
describe the phenomenon of interest; if it is a dynamical
phenomenon, then dynamical variables are associated with

Editor: Fran Lewitter, Whitehead Institute, United States of America

Citation: Aguda BD, Goryachev AB (2007) From pathways databases to network
models of switching behavior. PLoS Comput Biol 3(9): e152. doi:10.1371/journal.
pcbi.0030152
Copyright:  2007 Aguda and Goryachev. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Abbreviation: R point, restriction point
Baltazar D. Aguda is with the Mathematical Biosciences Institute, Ohio State
University, Columbus, Ohio, United States of America. Andrew B. Goryachev is with
the Centre for Integrative Systems Biology, School of Biological Sciences, University
of Edinburgh, United Kingdom.
* To whom correspondence should be addressed. E-mail: bdaguda@mbi.osu.edu

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doi:10.1371/journal.pcbi.0030152.g001

Figure 1. The Regulatory Network of the G1-S Transition in the

Mammalian Cell Cycle
Growth factors (GFs) trigger certain signaling cascades that lead to the
activation of cyclin D/CDK4 complexes. Active CDK4 phosphorylates
(thereby deactivating) the retinoblastoma protein (pRb) which inhibits
entry into S phase due mainly to inhibitory binding with E2F
transcription factors; these factors induce many of the genes required for
S phase (such as members of the pre-replication complex, cyclin E, cyclin
A, Cdc25A, etc.). Synthesis of cyclins E and A leads to activation of CDK2
which further phosphorylates (thereby deactivates) pRb. Another
transcription factor, namely Myc, also contributes to the G1-S transition,
but this proteins regulation is not shown. Arrows mean activate, and
hammerheads mean inhibit. The dashed arrows signify the totality of
gene expression steps (transcription and translation). Interactions
numbered 1 to 10 form a minimal model that can account for the R point
behavior.

doi:10.1371/journal.pcbi.0030152.g002

Figure 2. A Sharp Switch Is Expected from the Mutual-Activation and

Mutual-Inhibition Topology Involving CDK2
Also shown are the known detailed mechanistic steps corresponding to
the qNET shown in the upper panel (shaded grey). a refers to active,
and i to inactive. Note that the network in the lower figure uses the
chemists convention of representing reaction steps; also, dashed arrows
mean that the protein where the arrow originates from induces or
catalyzes the reaction step that the arrow points to.

are constants, and n could be an adjustable parameter of the

model.

Preliminary Network Models from Databases

the nodes, and rates of transitions from one variable to

another are associated with the edges.
An example of a network graph representation commonly
encountered in the molecular biology literature is shown in
Figure 1. The edges in the graph are all directed binary
interactions. The graph is referred to as a qualitative network
(qNET) because only the qualitative nature of the binary
interactions is depicted (an arrow means activates and a
hammerhead means inhibits).
If further details are known about the interactions, one can
transform the qNET into a mechanistic network model. An
example is illustrated in Figure 2 where the four qualitative
interactions among three nodes (upper panel) are shown to
correspond to seven mechanistic steps involving six proteins
or protein complexes (lower panel). In general, existing
online pathways databases contain mixtures of qualitative
and mechanistic interactions. In formulating the dynamical
equations of a network model having such mixtures of
interactions, standard chemical kinetic theory is used for the
rates of interactions with known mechanisms while
phenomenological equations (i.e., functional representations
of activation and inhibition) are used for the qualitative
interactions. An example of a phenomenological rate
equation representing the observation Y is inhibited by X is dY/
dt k1/(k2Xn), where X and Y are concentrations, k1 and k2
PLoS Computational Biology | www.ploscompbiol.org

In this and the next section, we illustrate how one can

extract a network model of the restriction point (R point) in the
mammalian cell cycle. The R point is a checkpoint in mid to
late G1 phase [6,7]. It is considered as the point of
commitment to replicate the DNA where it is sensed that
the prerequisites for cell cycle progression are satised (e.g.,
sufcient cell size and undamaged DNA). The modeling goal
is to explain the mechanistic and kinetic origins for an
observed switching behavior associated with the R point. This
switch pertains to the experimentally observed activation of
cyclin E/CDK2, as reported by Ekholm et al. [6] (see Figure 3).
In the model-extraction procedure discussed below, we will
refer to growth-factor signaling pathways as the processes
that induce the R point switch and to cyclin E/CDK2 as the
marker for the switch.
The rst step in building a network model is to identify the
nodes of the network. It would be easy to use literature
reviews written by specialists on the topic, but, as we
mentioned earlier, we start afresh by using information taken
from online databases. Since R point regulation is embedded
in the G1-S regulatory network, one may start by visiting the
Gene Ontology (GO) Web site (see Table 1) to obtain a list of
annotated genes. GOs classication and hierarchy of biological
processes can be used as a starting point for identifying the
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Table 1. A Few Major Pathway and Modeling Resources on the

Internet
Pathway

Modeling
Resources

URL

General Web Portal

Pathway Ontologies
and Notations

Pathguide
Gene Ontology

http://pathguide.org
http://www.geneontology.org

BioPAX
SBGN
KEGG
Reactome
GenMAPP
Biocarta
Pathway Interaction
Database
Biomodels
CellML

http://www.biopax.org
http://www.sbgn.org
http://www.genome.jp/kegg/
http://www.reactome.org
http://genmapp.org
http://biocarta.com
http://pid.nci.nih.gov/

Pathway Maps

Model Repositories

http://www.ebi.ac.uk/biomodels/
http://www.cellml.org

For a more comprehensive list, go to the Pathguide Web site http://pathguide.org.

doi:10.1371/journal.pcbi.0030152.t001

provided by Pathguide (see Table 1), which links to more than

200 Web sites containing a wide range of information from
pathway components (e.g., proteinprotein interactions) to
integrated pathway diagrams. A welcome activity in
bioinformatics is the ongoing development of standards for
pathway data representation by a biological pathways
exchange consortium called BioPAX (see Table 1).

doi:10.1371/journal.pcbi.0030152.g003

Figure 3. The Mammalian Cell Cycle Showing the G1, S, G2, and M
Phases along with the Predominant CyclinCDK Activities Associated
with Each Phase (Top Panel)
The lower panel shows the position of the R point (R) which subdivides
the G1 phase into G1-pm (post-mitosis) and G1-ps (pre-S-phase).
Quiescent or non-dividing cells have to be exposed to continuous
growth-factor stimulation up until the R point in order to commit to
entry into S-phase. After R and a finite induction period, cyclin E/CDK2
activity increases (shown by the dashed curve labelled E) as reported by
Ekholm et al. [6].

Extracting a Network Model

The following steps are sufcient to extract a network
model of the R point. (i) Start with an initial qNET large
enough to subsume the network model of interest. (ii)
Identify destabilizing cycles that involve the set of markers
and processes. The meaning of destabilizing cycle will be
given below. This step is required to nd an instability that is
assumed to be the cause of the switching behavior in the
activity of cyclin E/CDK2. (iii) A network model is formed
from the destabilizing cycles involving the marker and other
interactions encompassing the process involved. (iv) From the
network model, a kinetic model is generated using available
information on the mechanisms and rate expressions for the
interactions involved.
Step (i) requires knowledge of a set of biological markers
and processes associated with the phenomenon to be
modeled. For the R point, the initial qNET is given in Figure 1.
(To simplify the discussion, we have not included in Figure 1
many other nodes and interactions that can be found in
pathways databases.) As shown in Figure 3, the marker for
crossing the R point is taken to be cyclin E/CDK2 and the
process is that of growth-factor stimulation leading to the
activation of the marker. For step (ii), one has to make an
assumption as to what the arrows and hammerheads
mean. We will dene the interaction fXj Xig to mean
@[dxi/dt]/@xj . 0, i.e., Xj activates Xi because dxi/dt increases if
xj increases (the lowercase xs are the concentrations or
activities). Similarly, the interaction fXj j Xig means @[dxi/dt]/
@xj , 0, i.e., Xj inhibits Xi because dxi/dt decreases if xj
increases. These interpretations of the qualitative
interactions imply that a qNET graph corresponds to a

parts list of the G1-S molecular machinery. An example of a

GO search sequence, using the Amigo browser, is the
following: under the category of biological processes, click on
cellular process followed by cellular physiological process, cell cycle,
regulation of cell cycle, and cell cycle checkpoint. Another
recommended database is KEGG (Kyoto Encyclopedia of
Gene and Genomes, see Table 1). Within a network hierarchy
provided in KEGG BRITE, one nds (under KEGG pathway
maps) cellular processes, which links to KEGG PATHWAY,
containing manually curated pathway diagrams of the cell
cycle. For more details on the mechanisms, one can peruse
Reactome (see Table 1), which is a knowledgebase of human
biological pathways. Sources of network diagrams for specic
functional modules contributed by members of the research
community at large include GenMAPP, Biocarta, and PID (see
Table 1). We view these contributions as elements of a
growing library of pathway modules. One may nd redundancies,
inconsistencies, incompleteness, and other sources of
uncertainties in the contributed modules in these databases.
Nevertheless, it is useful to view these contributed modules as
preliminary models themselves for computational biologists
to investigate further.
A comprehensive Internet portal on pathway resources is
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From Network Models to Mathematical Models

Jacobian matrix M (whose element mij is equal to @[dxi/dt]/@xj.

Thus, a qNET graph gives only the algebraic signs of the
elements of M. From M one can perform a stability analysis of
the linearized network dynamics. The (stability) eigenvalues k
of M indicate whether the steady state of the system is locally
stable or not (it is unstable if any eigenvalue has a positive real
part). Note that only cycles in the qNET graph affect the
linear stability of the network; to prove this statement (see
also the appendix of [8]), one notes that the eigenvalues are
the roots of the characteristic polynomial, which can be
written as follows (for n 3 n matrix M):
detkI  M kn a1 kn1 a2 kn2 a3 kn3
::: an1 k an 0

Simulation of the network model of the R point required

the formulation of a system of coupled kinetic equations that
can then be solved to determine the dynamics of the biological
system [7]. The use of ordinary differential equations (ODEs)
with the R point model is possible due to mechanistic details
found in databases and the literature. However, when the
interactions in a network model are poorly dened,
simulation methods other than ODEs are employed.
Comprehensive reviews of these methods are already available
(for examples, see [1113]) and will not be repeated here.
These methods include stochastic simulations (applicable
when very few molecules are involved) and Boolean dynamic
simulations (applicable, for example, to a network of genes
that turn each other on or off). The translation of
network models to mathematical models is facilitated by the
use of XML-based languages such as SBML (systems biology
mark-up language) and CellML (see Table 1). Further
development of SBML [14] is under way to provide support for
storing and automatic generation of the graphical network
information necessary to describe a model in mathematical
terms. A parallel effort by the Systems Biology Graphics Notation
(SBGN) consortium (see Table 1) is the ongoing development
of a universal graphical notation for representation of various
kinds of interaction networks. Also of interest to the modeling
community is the creation of model repositories such as
Biomodels and CellML (see Table 1) for networks that have
matured into quantitative kinetic models.

where I is the n 3 n unit matrix. The coefcients ai in the

characteristic polynomial above can be expressed as follows:
X
a1
C1 i
i

a2

X
X
C1 iC1 j
C2 pq
pq

i;j

a3

X
X
C1 iC1 jC1 k
C1 tC2 pq
i; j;k

X
C3 vws; etc:

t; pq

vws

where Ck is a k-cycle in the qNET graph; examples of these

cycles for k 1, 2, 3 are: C1(i) mii, C2(pq) mpqmqp, and C3(vws)
mvwmwsmsv. The value of a Ck is also referred to as the
strength of that k-cycle. A cycle is said to be destabilizing if
increasing its strength leads to increasing the real part of at
least one eigenvalue toward the positive direction.
Carrying out step (ii) above on the network shown in
Figure 1, one only considers the destabilizing cycles that
involve the marker cyclin E/CDK2and these are the
following four positive loops (numbers are interaction
numbers): f3, 4, 10g, f6, 7g, f8, 9g, and f3, 5, 6, 10g. In step
(iii) abovewhich takes the aforementioned destabilizing
cycles, as well as the growth-factor signaling process
represented by steps 1 and 2one arrives at a network
model that is equivalent to the previously published model
of [7]. The network model is thus composed of edges
numbered 1 to 10 in Figure 1. There are several
destabilizing cycles in this network model. Of interest is the
instability due to phosphorylationdephosphorylation cycles
involved in the positive feedback interaction between
Cdc25A and cyclin E/CDK2 [7,9,10]. It was shown that the
switching point due to the positively coupled cycles is
reached only after the levels of the proteins involved have
grown above certain threshold values. Finally, step (iv) gives
rise to the detailed kinetic model of [7] that reproduces the
switching behavior of cyclin E/CDK2 shown in the
experiment depicted in Figure 3. The simulations presented
in [7] reproduce the induction period (when cyclin E/CDK2
activity is very low) after the R point, followed by the sudden
increase in cyclin E/CDK2 activity; more importantly, the
model simulations also show that cutting off growth-factor
stimulation after the R point does not prevent the activation
of CDK2.
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Concluding Remarks
The main goal of this article is to illustrate the idea that
network models can be extracted from pathways databases in
a systematic way. Using a specic biological phenomenon,
namely the R point in the cell cycle, the modeling task is to
explain the origin of the switching behavior of a protein
marker when a quiescent cell is exposed to sufcient growthfactor stimulation. A large network of molecular interactions
and signaling pathways is integrated from various pathways
databases. Despite the lack of quantitative kinetic parameters
associated with almost all of the interactions, we
demonstrated that the form of qualitative network analysis
described here can identify key feedback cycles in the
network with potential for instability (the ultimate cause of
the switching behavior). The set of these cycles is the basis for
the reduced qualitative network model. Computer
simulations using the nal kinetic model [7]which includes
known mechanistic detailsvalidate the prediction of a
switching behavior by the model. For another detailed
example of the application of the modeling approach
discussed in this paper, the reader is referred to a recent work
of Wee and Aguda [15] on the network of interactions
between the tumor suppressor protein p53 and the
oncoprotein Akt; here, the predicted switching behavior
between pro-apoptotic and pro-survival cellular pathways is
based on the presence of destabilizing cycles in the
network. &

Acknowledgments
The support of Professor Avner Friedman and the US National
Science Foundation during BDAs visit at the Mathematical
Biosciences Institute is gratefully acknowledged. ABG is supported by
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6. Ekholm SV, Zickert P, Reed SI, Zetterberg A (2001) Accumulation of cyclin

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10. Aguda BD (1999) A quantitative analysis of the kinetics of the G2 DNA
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an Research Councils United Kingdom Fellowship at the University

of Edinburgh.
Author contributions. Both authors contributed to writing the
paper.
Funding. The authors received no specic funding for this article.
Competing interests. The authors have declared that no competing
interests exist.
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